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Santa Fe de la Vera Cruz, Argentina

Ceaglio N.,National University of Santa | Gugliotta A.,National University of Santa | Tardivo M.B.,Zelltek SA | Cravero D.,National University of Santa | And 3 more authors.
Journal of Biotechnology | Year: 2016

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) β-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity. © 2016 Elsevier B.V. Source


Amadeo I.,Zelltek SA | Amadeo I.,National University of Santa | Mauro L.V.,Zelltek SA | Orti E.,Zelltek SA | And 2 more authors.
Biotechnology Progress | Year: 2011

A typical chromatographic purification step has numerous operating parameters that can impact its performance. As it is not feasible to evaluate the influence of each one, the current practice in biopharmaceutical industry is to apply risk analysis approach to identify process parameters that should be examined during process characterization. Once these parameters are identified, a response surface study can be run to help understand the relationship between critical inputs and outputs. We performed a study comprising optimization and robustness determination for a Blue-Sepharose purification step of rhEPO, a well-known therapeutic glycoprotein. Initially, risk analysis was fulfilled to identify key parameters. A small-scale model was created and qualified before its use in experimental studies, given by a Box-Behnken design with three factors. This method proved to be a very useful tool in bioprocess validation studies in which many input variables can affect product quality and safety. © 2011 American Institute of Chemical Engineers (AIChE). Source


Mattio M.,National University of Santa | Ceaglio N.,National University of Santa | Oggero M.,National University of Santa | Perotti N.,National University of Santa | And 5 more authors.
Biotechnology Progress | Year: 2011

Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side-effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms -designated as neuroepoetin (rhNEPO)- was purified to homogeneity from the supernatant of a CHO-producing cell line by a four-step chromatographic procedure. This simple and single process allowed us to prepare two EPO derivatives with distinct therapeutic expectations: the hematopoietic version and a minimally hematopoietic, but mainly in vitro cytoprotective, alternative. Further biological characterization showed that the in vivo erythropoietic activity of rhNEPO was 25-times lower than that of rhEPO. Interestingly, using different in vitro cytoprotective assays we found that this molecule exerts cytoprotection equivalent to, or better than, that of rhEPO in cells of neural phenotype. Furthermore, despite its shorter plasma half-life, rhNEPO was rapidly absorbed and promptly detected in the cerebrospinal fluid after intravenous administration in rats (5 min postinjection, in comparison with 30 min for rhEPO). Therefore, our results support the study of neuroepoetin as a potential drug for the treatment of neurological diseases, combining high cytoprotective activity with reduced hematological side-effects. © 2011 American Institute of Chemical Engineers (AIChE). Source

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